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An engineered transforming growth factor ? (TGF-?) monomer that functions as a dominant negative to block TGF-? signaling.

ABSTRACT: The transforming growth factor ? isoforms, TGF-?1, -?2, and -?3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-? pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-?s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-? monomer, lacking the heel helix, a structural motif essential for binding the TGF-? type I receptor (T?RI) but dispensable for binding the other receptor required for TGF-? signaling, the TGF-? type II receptor (T?RII), as an alternative therapeutic modality for blocking TGF-? signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-? monomers and bound T?RII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-? signaling with a Ki of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for T?RII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit T?RI, enabled it to bind endogenous T?RII but prevented it from binding and recruiting T?RI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-? signaling and may inform similar modifications of other TGF-? family members.

SUBMITTER: Kim SK

PROVIDER: S-EPMC5409485 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling.

Kim Sun Kyung SK Barron Lindsey L Hinck Cynthia S CS Petrunak Elyse M EM Cano Kristin E KE Thangirala Avinash A Iskra Brian B Brothers Molly M Vonberg Machell M Leal Belinda B Richter Blair B Kodali Ravindra R Taylor Alexander B AB Du Shoucheng S Barnes Christopher O CO Sulea Traian T Calero Guillermo G Hart P John PJ Hart Matthew J MJ Demeler Borries B Hinck Andrew P AP

The Journal of biological chemistry 20170222 17

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, w ...[more]

PMID: 28228478

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Project description:BackgroundChronic fatigue syndrome (CFS) is a prevalent and disabling condition among adolescent. The disease mechanisms are unknown. Previous studies have suggested elevated plasma levels of several cytokines, but a recent meta-analysis of 38 articles found that of 77 different cytokines measured in plasma, transforming growth factor beta (TGF-β) was the only one that was elevated in patients compared to controls in a sufficient number of articles. In the present study we therefore compared the plasma levels of the three TGF-β isoforms in adolescent CFS patients and healthy controls. In addition, the study explored associations between TGF-β levels, neuroendocrine markers, clinical markers and differentially expressed genes within the CFS group.MethodsCFS patients aged 12-18 years (n = 120) were recruited nation-wide to a single referral center as part of the NorCAPITAL project (ClinicalTrials ID: NCT01040429). A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls (n = 68) were recruited from local schools. The three isoforms of TGF-β (TGF-β1, TGF-β2, TGF-β3) were assayed using multiplex technology. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings. Whole blood gene expression was assessed by RNA sequencing in a subgroup of patients (n = 29) and controls (n = 18).ResultsPlasma levels of all three isoforms of TGF-β were equal in the CFS patients and the healthy controls. Subgrouping according to the Fukuda and Canada 2003 criteria of CFS did not reveal differential results. Within the CFS group, all isoforms of TGF-β were associated with plasma cortisol, urine norepinephrine and urine epinephrine, and this association pattern was related to fatigue score. Also, TGF-β3 was related to expression of the B cell annotated genes TNFRSF13C and CXCR5.ConclusionsPlasma levels of all TGF-β isoforms were not altered in adolescent CFS. However, the TGF-β isoforms were associated with neuroendocrine markers, an association related to fatigue score. Furthermore, TGF-β3 might partly mediate an association between plasma cortisol and B cell gene expression. Trial registration Clinical Trials NCT01040429.

Project description:Appropriate growth and development of the endometrium across the menstrual cycle is key for a woman's quality of life and reproductive well-being. Recurrent pregnancy loss (RPL) and heavy menstrual bleeding (HMB) affect a significant proportion of the female population worldwide. These endometrial pathologies have a significant impact on a woman's quality of life as well as placing a high economic burden on a country's health service. An underlying cause for both conditions is unknown in approximately 50% of cases. Previous research has demonstrated that aberrant endometrial vascular maturation is associated with both RPL and HMB, where it is increased in RPL but reduced in HMB. TGFβ1 is one of the key growth factors that regulate vascular maturation, by inducing phenotypic switching of vascular smooth muscle cells (VSMCs) from a synthetic phenotype to a more contractile one. Our previous data demonstrated an increase in TGFβ1 in the endometrium of RPL, while others have shown a decrease in women with HMB. However, TGFβ1 bioavailability is tightly controlled, and we therefore sought to perform an extensive immunohistochemical analysis of different components in the pathway in the endometrium of normal controls, women with HMB or RPL. In addition, two in vitro models were used to examine the role of TGFβ1 in endometrial vascular maturation and endothelial cell (EC):VSMC association. Taken all together, the immunohistochemical data suggest a decrease in bioavailability, receptor binding capacity, and signaling in the endometrium of women with HMB compared with controls. In contrast, there is an increase in the bioavailability of active TGFβ1 in the endometrium of women with RPL compared with controls. Endometrial explants cultured in TGFβ1 had an increase in the number of vessels associated with contractile VSMC markers, although the total number of vessels did not increase. In addition, TGFβ1 increased EC:VSMC association in an in vitro model. In conclusion, TGFβ1 is a key regulator of endometrial vascular maturation and could be considered as a therapeutic target for women suffering from HMB and/or RPL.

Dataset Information

An engineered transforming growth factor ? (TGF-?) monomer that functions as a dominant negative to block TGF-? signaling.

Publications

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling.

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